The long-term objective of this work is the continued analysis of mechanisms involved in the cardioprotective actions of volatile anesthetics such as isoflurane. Cardioprotection produced by these drugs is well documented across diverse species including rodents, large animals and humans, and mimics ischemic preconditioning in that a prior exposure to the volatile agent continues to be protective after the anesthetic has been eliminated, a phenomenon we termed "anesthetic preconditioning" (ARC). Project I will extend key findings of the previous funding cycle to investigate the global hypothesis that administration of volatile anesthetics such as isoflurane causes a cascade of events including activation of the hypoxia inducible factor la-vascular endothelial growth factor-nitric oxide (HIF1a-VEGF-NO) axis leading to ARC. Specific Aim I will test hypotheses related to how isoflurane regulates myocardial concentrations of HIF1a including actions on intracellular kinases, reactive oxygen species and/or the chaperone protein heat shock protein 90 (HSP90). Enhanced synthesis versus reduced ubiquination mechanisms elicited by isoflurane will be examined. Specific Aim II will test hypotheses related to the contribution of VEGF as an intermediate cardioprotective protein in ARC. Whether VEGF is a mediator of alterations in sarcKATp channel sensitivity and mitochondrial permeability transition by isoflurane will be explored. The importance of vascular endothelium in the protection of cardiomyocytes against hypoxia+reoxygenation injury by isoflurane will be established. Specific Aim III will test hypotheses related to the role and mechanism of action of NO in ARC. The NO synthase (NOS) isoform(s) involved in ARC will be delineated. The effect of NOS inhibition on isoflurane-induced changes in mitochondrial function, mitochondrial proteome, and NO feedback on HIF1a will be determined. NO feedback on HIF1a will be examined as the basis for the memory of ARC. A combination of in vivo rat and mouse models of cardioprotection and in vitro endothelial cell and cardiomyocyte cultures will be used to address the Specific Aims. Molecular and biochemical analyses related to the Specific Aims will be conducted with support from the Molecular and Biochemical and Proteomics Cores. The actions of reactive oxygen species and NO on mitochondrial function and effects of HSP90, an intracellular protein chaperone on the cardioprotection elicited by isoflurane will be studied in collaboration with investigators from Project II and III, respectively. Lay description: Cardiovascular disease is a significant problem in our society, and it is important to define new therapeutic strategies, including optimal delivery of anesthesia to patients with coronary artery disease. Protection against ischemia and reperfusion injury by drugs including the volatile anesthetic isoflurane is a complex process involving multiple intracellular mediators which will be defined by the proposed research.